1. Field of the Invention
This invention relates to novel diphenylhydantoin derivatives pertaining to binding assays for detecting diphenylhydantoin and its salt forms in liquid media, such as serum, saliva, and cerebrospinal fluid. Such derivatives include labeled diphenylhydantoin conjugates directly used in carrying out such assays. Also described are novel immunogen conjugates for preparing diphenylhydantoin-specific antibodies according to conventional techniques. Further, there are described novel intermediates useful in the synthesis of such labeled conjugates and such immunogen conjugates.
Diphenylhydantoin (5,5-diphenyl-2,4-imidazolidinedione), also known by the generic name phenytoin and by various trademarks including Dilantin, is an anti-convulsant drug useful in the management of epilepsy, having the formula: ##STR1## wherein .phi. represents phenyl [cf. The Merck Index, 9th edition, p. 952 (1976)].
Like most anti-convulsants, diphenylhydantoin possesses a low therapeutic index and has clearly separable ranges of drug concentration in blood in which it is ineffective, therapeutic, or toxic, respectively. At sufficiently high concentration, the drug is potentially toxic and is eliminated from the body at a rate which can vary within a wide range from individual to individual so that the same dose can yield an ineffective, a therapeutic, or a toxic concentration depending on the subject. Administration of usual doses of diphenylhydantoin produces serum concentrations from 5 up to 50 micrograms/milliliter (.mu.g/ml) due to differences in the rate of hepatic metabolism of the drug. However, in almost all patients, the therapeutic range of serum concentration lies between 10 and 20 .mu.g/ml whereas toxic signs of nystagmus, ataxia and mental changes almost invariably appear at blood levels over 20 .mu.g/ml. With usual doses of diphenylhydantoin, some patients will have serum concentrations outside the therapeutic range, indicating the need for dosage adjustment.
2. Brief Description of the Prior Art
Over the years, many varied assay techniques have evolved for the monitoring of diphenylhydantoin levels in serum, including spectrophotometry, thin-layer chromatography, high-pressure liquid chromatography, and immunological methods. The state-of-the-art immunoassay techniques comprise the radioimmunoassay methods of Tigelaar et al, Clin. Chim. Acta 43:231-241 (1973) and Cook et al as described in Quantitative Analytic Studies in Epilepsy, ed. Kellaway et al, Raven Press (New York, 1976), pp. 39-58; and the enzyme immunoassay technique described in U.S. Pat. Nos. 3,817,837 and 3,905,871.
A specific binding assay for detecting ligands, including drugs such as diphenylhydantoin, employing an enzyme-cleavable .beta.-galactosyl-umbelliferone residue as label is described in pending U.S. patent application Ser. No. 886,094, filed Mar. 13, 1978, assigned to the instant assignee.
Methods of synthesizing diphenylhydantoin immunogen conjugates and using same to obtain specific antibodies are described by Tigelaar et al, supra and Cook et al, Res. Commun. Chem. Pathol. and Pharmacol. 5:767-774 (1973) and in U.S. Pat. No. 3,995,021 and French Pat. No. 2,276,317.